Kerstin Buettner wrote- >I am stuck with a very basic problem. I am trying to separate dead from >viable cells. People in my lab have always done this with 10µgPI/ml plus >RNase for 20min room temperature. I have tried two other protocols: PI >2µg/ml 5min on ice and AAD 1µg/ml 30min on ice. Every protocol yields >different counts of dead cells - from 16 +/-3% (AAD), 22+/-7% (PI) to >27+/-5% (PI combined with RNase). >Is this a probem of concentration - are DNA-Dyes titreable like an antibody? >Is it a question of temperature or time? > >We do not understand this, because we always thought that a dead cell is >dead, has permeabilised membranes and therfore takes up the dye - not >depending on concentration. We always thought that higher concentration >only gives you a better separation of peaks. It is true that dead cells with permeabilized membranes should take up otherwise impermeant nucleic acid dyes such as 7-AAD and PI. Note that propidium (PI) and ethidium (EB) should not be used interchangeably; ethidium has only a single positive charge, and can get into intact cells, especially at relatively high pH, but is pumped out. Propidium has two positive charges and generally does not get in. TO-PRO-1 and TO-PRO-3 also have two positive charges, and, in my limited experience (unpublished) using PI (488 nm excitation) and TO-PRO-3 (633 nm excitation) together, cells that take up one dye take up the other. However, my colleagues and I have also observed (Novo et al, Antimicrob Agents Chemother 2000, 44:827-834 [should be on PubMed]; Shapiro, Cytometry 2001, 43:223-226 and Shapiro, J Microbiol Methods 2000, 42:3-16 [this is in a special issue on Microbial Analysis at the Single Cell Level, and it and all other articles are available free at http://www.elsevier.com/locate/jmicmeth]) that, under some conditions, bacteria that maintain their membrane potentials, and which therefore have intact membranes, take up PI or TO-PRO-3. The "permeabilization" that permits PI uptake is usually conceived as a big hole in the membrane, which should also allow equilibration of ion concentrations across the membrane, reducing membrane potential to zero. This is clearly not the situation just described; the PI or TO-PRO-3 would have to get in via some more specific mechanism, e.g., a transport protein, and I have heard that such transport proteins have been described. Suffice to say that the "membrane permeability" which is taken as evidence of nonviability when dye exclusion tests are used may be substantially more complex than was originally thought. At a simpler level, there is the possibility that the different experimental conditions you use could affect the development of membrane permeability in dying (e.g., apoptotic) cells; while early apoptotic cells exclude PI and can be identified only by other criteria (Annexin V binding, mitochondrial membrane potential changes), these cells eventually end up permeable to PI. Along the way, you may see weak staining, so you have to decide what is "positive". I don't think enough has been published on this topic; if the numbers you report hold up in a repeat experiment, you should put together a manuscript. >How do I know which concentration is the right one for my AAD? That would depend on whether you can resolve your initial problem or not. Under normal circumstances, you shouldn't need to titrate nucleic acid dyes for dye exclusion tests. >And there ist still another problem with PI: If I stay with PI - is it more >accurate to compensate PI out of FL2 (which in fact means 99,9% >compensation) or is it better to leave the PI signal where it is, since >the dead cells are gated out anyway before the PE stained cells in FL2 are >analyzed? I'd leave the signal as is, since it puts the dead (or permeabilized) cells off scale and lets you gate them out. -Howard
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